EP0577523B1 - Fragment de l'ADN génomique de Streptococcus pneumoniae, sonde d'hybridation, amorce d'amplification, réactif et procédé de détection de Streptococcus pneumoniae - Google Patents
Fragment de l'ADN génomique de Streptococcus pneumoniae, sonde d'hybridation, amorce d'amplification, réactif et procédé de détection de Streptococcus pneumoniae Download PDFInfo
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- EP0577523B1 EP0577523B1 EP93420061A EP93420061A EP0577523B1 EP 0577523 B1 EP0577523 B1 EP 0577523B1 EP 93420061 A EP93420061 A EP 93420061A EP 93420061 A EP93420061 A EP 93420061A EP 0577523 B1 EP0577523 B1 EP 0577523B1
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- streptococcus pneumoniae
- sequence
- nucleotide sequence
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Definitions
- the present invention relates to a fragment of the genomic DNA of the bacterium Streptococcus pneumoniae, a probe capable of hybridizing specifically with the genomic DNA of Streptococcus pneumoniae, a primer to specifically amplify the genomic DNA of Streptococcus pneumoniae, a reagent and a method for selectively detecting, in a biological sample, said bacterium, implemented with the probe of the invention.
- various sub-fragments were prepared and used as a probe for hybridization of the genomic DNA of Streptococcus pneumoniae .
- the most commonly used probes are for the hexB gene , the hexB-S 7 fragment obtained by the action of restriction enzymes HindIII-Bgl / II (from nucleotide 1321 to nucleotide 1776), of 455 nucleotides and for the friend operon , the ami-S 2 fragment, obtained by the action of BamHI-EcoRI restriction enzymes (from nucleotide 2419 to nucleotide 3564), of 1145 nucleotides.
- This probe was tested on 44 strains of Streptococci among which 27 were identified as strains of atypical Streptococci and the other 17 as strains of Streptococcus viridans , using standard characterization tests.
- the present invention aims to solve the aforementioned problems of selective detection of Streptococcus pneumoniae, in particular encountered in medical bacteriology.
- the first subject of the invention is a single-stranded fragment of the genomic DNA of Streptococcus pneumoniae comprising at least one nucleotide sequence having at least 70% homology with at least one nucleotide sequence chosen from the nucleotide sequences SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, shown at the end of the description, and their respective complementary sequences.
- complementary sequence is meant any sequence that completely hybridizes with the sequence shown.
- the nucleotide sequence of the fragment according to the invention has at least 85% homology with at least one of said nucleotide sequences.
- a second object of the invention directly implements said fragment and consists of a probe capable of hybridizing specifically with the genomic DNA of Streptococcus pneumoniae, said probe comprising a nucleotide sequence having at least 70% homology with at least part of a consensus sequence of the genomic DNA of Streptococcus pneumoniae, this consensus sequence being chosen from the nucleotide sequences SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4 and their respective complementary sequences.
- the nucleotide sequence of the probe of the invention has at least 85% homology with at least part of said consensus sequence.
- the probe of the invention advantageously comprises at least 12 nucleotides.
- the probe of the invention comprises the nucleotide sequence, SEQ ID NO 3.
- SEQ ID NO 3 When it understands SEQ ID NO 3, it can be flanked at its 5 'end of the nucleotide sequence SEQ ID NO 2 and / or at its 3 'end of the sequence nucleotide SEQ ID NO 4.
- the nucleotide sequence SEQ ID NO 3, can be repeated and it is advantageously repeated four times contiguously.
- a probe can have a shorter nucleotide sequence and be chosen from the sequences SEQ ID NO 5, SEQ ID NO 6 and SEQ ID NO 7, presented at the end of the description.
- the labeling of the probe does not influence its specificity vis-à-vis the genomic DNA of Streptococcus pneumoniae and an appropriate marker is preferably chosen from radioactive isotopes, enzymes chosen from horseradish peroxidase and alkaline phosphatase and those capable of hydrolyzing a chromogenic, fluorigenic or luminescent substrate, chromophoric chemical compounds, chromogenic, fluorigenic or luminescent compounds, nucleotide base analogs and biotin.
- an appropriate marker is preferably chosen from radioactive isotopes, enzymes chosen from horseradish peroxidase and alkaline phosphatase and those capable of hydrolyzing a chromogenic, fluorigenic or luminescent substrate, chromophoric chemical compounds, chromogenic, fluorigenic or luminescent compounds, nucleotide base analogs and biotin.
- the molecular structure of the latter is chemically modified.
- Appropriate chemical modifications which make it possible to improve the resistance with respect to an enzymatic degradation, in particular due to nucleases, and to further increase the yield of hybridization, of course does not affect the sequence bases. Examples are the introduction between at least two nucleotides of a group chosen from esters of diphosphate, alkyl- and acryl-phosphonate and phosphorothioate or the replacement of at least one deoxyribose by a polyamide.
- a third object of the invention is a primer (or "primer") for the specific polymerization of the genomic DNA of Streptococcus pneumoniae , with a view to carrying out an amplification of the latter.
- This primer comprises a nucleotide sequence having at least 70% homology with at least part of a consensus sequence of the genomic DNA of Streptococcus pneumoniae , this consensus sequence being chosen from the nucleotide sequences SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4 and their respective complementary sequences.
- nucleotide sequence of a primer of the invention is chosen from the sequences SEQ ID NO 8 to SEQ ID NO 21, shown at the end of description.
- primers including at least one primer of the invention.
- said pair is advantageously chosen from the probe pairs constituted by a probe having the nucleotide sequence SEQ ID NO 8 and one probe corresponding to any of the sequences nucleotides SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 19 and SEQ ID NO 21 and among the probe couples constituted by a probe responding to the nucleotide sequence SEQ ID NO 10 and a probe responding any of the nucleotide sequences SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 19 and SEQ ID NO 21.
- a fourth subject of the invention is a reagent to selectively detect Streptococcus pneumoniae in a biological sample, applying a probe of the invention as described above.
- the reagent of the invention comprises a capture probe and a detection probe, presenting the characteristics of a probe of the invention, the probe of detection being notably marked by means of one of the markers described above.
- any polynucleotide is called a capture probe fixed on a macromolecular support capable of hybridizing with a part (or capture zone) of a nucleic acid to be detected in a sample (the target), in particular DNA.
- the capture probe can be a fragment natural nucleic acid (especially DNA), a natural or synthetic oligonucleotide or fragment synthetic nucleic acid (especially DNA) unmodified or comprising one or more modified bases such as inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, diamino-2,6-purine, bromo-5-deoxyuridine or any another modified base allowing hybridization.
- modified bases such as inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, diamino-2,6-purine, bromo-5-deoxyuridine or any another modified base allowing hybridization.
- the detection probe is a probe capable of hybridize to part of the target (detection zone) which meets the definition above and is marked by any appropriate marker and in particular chosen from markers cited above.
- the probe of the invention is in liquid medium fixed on a solid support, directly or indirectly.
- Said support is in any form suitable such as tube, cone, well, plate microtitration, sheet, soluble polymer. It is made of a natural, synthetic, modified material chemically or not and is, depending on the technique used, chosen from polystyrenes, styrenebutadiene copolymers, styrene-butadiene copolymers in admixture with polystyrenes, polypropylenes, polycarbonates, polystyrene-acrylonitrile copolymers, styrene-methyl methyl methacrylate copolymers, among Nylon and natural synthetic fibers, among polysaccharides and cellulose derivatives.
- the reagent of the invention may contain at least one primer as described above, to allow an amplification technique to be used before the selective detection of Streptococcus pneumoniae, and preferably it contains a pair of primers according to the invention.
- the last object of the present invention is a method for the selective detection of Streptococcus pneumoniae in a biological sample, consisting in exposing the genomic DNA of the bacteria contained in said sample, in the form of single-stranded fragments, to a probe of the invention, then detecting the hybridization zones with said probe.
- the method consists in exposing the genomic DNA of the bacteria of the sample beforehand to a capture probe of the invention, on which the genomic DNA of Streptococcus pneumoniae will specifically bind , then exposing the DNA attached to a detection probe of the invention.
- the method advantageously comprises a step of amplifying the genomic DNA of Streptococcus pneumonia, in the presence of a suitable enzymatic system and at least one primer and in particular a pair of primers of the invention, prior to the detection step of Streptococcus pneumoniae.
- a particular sequence responsible for these multiple hybridizations has been located on this fragment. It is a nucleotide sequence located in the downstream region, in the 5 '--->3' direction, of the mmsA gene. This sequence obtained by the action of the restriction enzymes HpaI and PvuII, has 340 base pairs, according to SEQ ID NO 1 given at the end of the description. The complete nucleotide sequence of the two complementary strands of this fragment was determined by the chain termination method (according to SANGER et al., Proc. Natl. Acad. Sci. USA, 1977, 74 , 5463-5467) from matrices single-stranded DNA from phage M13. We then highlighted, within this sequence, the existence of a sequence of 45 nucleotides called boxB , directly repeated 4 times.
- boxB sequence was then found by comparison of sequences in regions upstream of the hexB, comA, lytA, ply, SI, SII genes. It was found that these copies of boxB could be flanked in 5 ′ and 3 ′ of sequences of around fifty nucleotides, also conserved, called respectively boxA and boxC .
- A, B, C we determined by alignment of the different nucleotide sequences of the regions of hexB, comA, lytA, ply, SI, SII , a consensus sequence corresponding to the most frequently found nucleotide sequence in these regions.
- the consensus sequences SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4 correspond to the boxes A, B, C respectively .
- Chromosome organization and localization Chromosome organization and localization :
- these alignments made it possible to define the sequence and the location of three oligodeoxyribonucleotides indicated at the end of the description according to the references respectively SEQ ID NO 5, SEQ ID NO 6 and SEQ ID NO 7.
- One of these oligonucleotides is derived alignment of copies of boxA (SEQ ID NO 5).
- the second oligodeoxyribonucleotide (SEQ ID NO 6) is derived from the alignment of copies of boxB
- a third (SEQ ID NO 7) is derived from the alignment of copies of boxC .
- oligodeoxyribonucleotides as well as any other consensus sequence established on the basis of data from box A, box B and box C or any sequence exhibiting at least 70% homology with one of the consensus sequences can be used as a probe specific for genomic DNA. Streptococcus pneumoniae.
- Streptococcus oralis which comprises various strains previously classified S. sanguis II, S. mitior, S. viridans, and S. mitis are the two closest species of Streptococcus pneumoniae .
- the strain of Streptococcus oralis NCTC 11427 used in this study is the type strain (Kilpper-Bälz et al., 1985, and Coykendall, AL 1989, Classification and identification of Viridans Streptococci. Clin. Microbiol. Rev. 2, 315-328, Kilian, M., Mikkelsen, L., and Henrichsen, H. 1989, Taxonomic study of Viridans Strepotococci: description of Streptococcus gordonii sp. Nov.
- Streptococcus sanguis White and Niven 1946
- Streptococcus oralis Bridge and Sneath 1982
- Streptococcus mitis Andrewes and Horder, 1906), Int. J. Sys. Bact. 39, 471-484. It is moreover from this strain that the DNA fragment which constitutes a specific probe for Streptococcus oralis was isolated (Schmidhuber et al., 1988). Streptococcus mitis is represented by two clinical isolates.
- Streptococcus gordonii newly created species (Kilian et al., 1989) which includes a number of previously classified strains S. sanguis II, represented by the strain OB11 (ex S. sanguis Challis) (Kilian et al., 1989, Haisman and Jenkinson, 1991), as well as S. sanguis which is not represented in this study can be considered, according to the DNA-DNA hybridization results, as less close to Streptococcus pneumoniae than Streptococcus oralis Streptococcus mitis .
- the chromosomal DNA of the different strains of streptococci was prepared by the technique described by Fenoll et al. (1990).
- the enzymatic digestion by the enzyme Pstl as well as the electrophoresis on agarose gel and the transfers on charged nylon membrane (Biodyne B, provenance PALL) were carried out under the conditions described by Maniatis, T., Fristsch, EF, and Sambrook , (1982). Molecular cloning; A laboratory manual (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory).
- the oligodeoxyribonucleotide was labeled in 5 ′ by the DNA kinase of bacteriophage T4 (from Bethesda Research Laboratory) with ATP ⁇ 32 P (at 3000 Ci / mM). 50 ⁇ l of labeled oligonucleotide solution (1x10 7 cpm for approximately 2.5 picomoles) were introduced into a hybridization buffer [6xSSC (Saline Sodium Citrate), 10x Denhardt's, 0.1% SDS (Na dodecyl sulfate) ], 50 mg / ml (salmon sperm DNA, 1% boehringer blocking reagent) (Maniatis et al., 1982).
- Hybridization was carried out at 40 ° C (probe of SEQ ID NO 6) or 48 ° C (probe of SEQ ID NO 7), for approximately 15 hours. After two brief washes in a solution of 6xSSC, 0.1% SDS, at room temperature, the membrane was brought into contact with an X-ray film which was exposed for 3 to 36 hours at -70 ° C.
- oligonucleotides SEQ ID NO 6 and SEQ ID NO 7 as a probe.
- Chromosomal DNA from Streptococcus pneumoniae and other streptococci including Streptococcus oralis and Streptococcus gordonii two of the species closest to this bacteria were digested with the restriction enzyme Pstl , separated by agarose gel electrophoresis, then transferred on Nylon membrane.
- the 32 P-labeled oligonucleotide was brought into contact with this membrane, under standard hybridization conditions.
- the hybridization results show very important hybridization signals obtained with the DNA of Streptococcus pneumoniae , while they do not exist with the DNA of Streptococcus oralis , the closest species to Streptococcus pneumoniae , as well as with the Of Streptococcus gordonii , clinical isolates classified as Streptococcus sanguis , and one of the clinical isolates classified as Streptococcus mitis .
- STEP 4 Identification of Streptococcus pneumoniae by direct hybridization on colonies using a non-radioactive and semi-automated detection system described in French patent FR2663040.
- the extraction of total DNA from the colonies is carried out as follows. A bacterial colony standardized to a 10 9 bacteria inoculum is taken up in 400 ⁇ l of a 0.1M sodium citrate solution containing 0.85 g of sodium chloride. 40 ⁇ l of sodium deoxycholate detergent (1%) are added. After incubation for 5 minutes at room temperature, 4 phenolchloroform extractions are carried out (Maniatis et al. 1982). The DNA is precipitated with ethanol. The pellet is taken up in 100 ⁇ l of sodium citrate buffer. this solution is sonicated with a 60W sonicator (Bioblock Company under ref. C72442) using a "cuphorn" type probe (Bioblock Company under ref. C72438) in order to obtain a population of fragments of majority size of 1 Kilobase
- a sample corresponding to 10 8 bacteria in 10 ⁇ l is then identified by hybridization according to the following protocol.
- a microtiter plate (Trade name Nunc 439454) is deposited a solution of the oligonucleotide capture probe (probe SEQ ID NO 6) at 1 ng / ⁇ l in PBS 1X (0.15 M NaCl, 0.05 M sodium phosphate, pH 7.0 ).
- the plate is incubated for 2 h at 37 ° C. and then washed 3 times with 300 ⁇ l of PBST (PSB + detergent of TWEEN brand from the company MERCK).
- the target consists of 10 .mu.l of the total DNA is sonicated mixed with 70 ⁇ l of PBS salmon buffer [3 ⁇ PBS + salmon sperm DNA 10 ⁇ l / ml (Sigma Company under item no. D9156)] and 10 .mu.l of 2N soda. The whole is neutralized 5 minutes later, by the addition of 10 ⁇ l of 2N acetic acid. The whole is added to the well in addition to 50 .mu.l of a solution of labeled detection probe conjugated with peroxidase according to SEQ ID NO 7 at a concentration of 0.1 ng / ⁇ l in a horse PBS buffer [3 ⁇ PBS + 10% horse serum, (Instituteaueux SA ref. 55842)].
- the plate is incubated 1 hour at 37 ° C and washed with 3X 300 ⁇ l of PBS Tween [PBS1X + 0.5% Tween 20 (Merck ref 822184)].
- OPD substrate ortho-phenylenediamine from Cambridge Medical Biotechnology ref / 456
- a specific buffer 0.055 M citric acid, 0.1 M Na 2 HPO 4 , pH 4.93
- H 2 O 2 to 30 volumes in 1/1000, are added per well.
- the enzymatic activity is blocked by 100 ⁇ l of 1N H 2 SO 4 and the reading is carried out on a microplate reader of the registered trademark Axia Microreader (Company bioMérieux SA) at 492 nm.
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Description
a1-a11 : Isolats clinique de Streptococcus pneumoniae appartenant à différents sérotypes.
c : "Streptocoques atypiques" provenant d'isolats cliniques.
- Toutes les souches de Streptococcus pneumoniae de la série a) donnent des signaux très visibles qui'sont proportionnels à la concentration considérée.
- Les streptocoques atypiques de la série C, C91, C120, C108, C92, C188, C139, C155, C184, C115, C65 et C174 donnent les mêmes signaux que ceux de la série a) et pourraient alors être classifiés, selon ce test, dans l'espèce des Streptococcus pneumoniae.
- Toutes les souches de Streptococcus oralis de la série b), à l'exception de la souche b5, la souche Streptococcus mitis b11 et les streptocoques atypiques de la série C, C185, C160, C85 donnent un signal visible pour la concentration de 100ng, l'intensité du signal étant environ 10 fois plus faible que celle obtenue pour les Streptococcus pneumoniae à la même concentration.
- la sonde utilisée est un fragment de 650 paires de bases et sa production industrielle n'est en conséquence pas aisée,
- et surtout, la spécificité de cette sonde est exclusivement liée à,la présence du gène lyt A, dont la copie est unique dans l'ADN génomique de Streptococcus pneumoniae, donc elle ne pourra pas détecter ou identifier une souche de S. pneumoniae dont le gène lyt A a été délété; en outre, ce nombre réduit de copies est désavantageux pour la détection par hybridation directe et pour l'amplification de l'ADN cible.
- figure 2, permet de localiser, au voisinage de différents gènes de Streptococcus pneumoniae, les sénucléotidiques répétées appelées respectivement boiteA, boíteB, boíteC
- figure 3 illustre la détermination des séquences consensus respectivement SEQ ID NO 2, SEQ ID NO 3 et SEQ ID NO 4 par alignement des différentes séquences répétées au voisinage de 5 gènes de Streptococcus pneumoniae.
- comA : 7'
- hexB :10-11'
- lytA : 21-24'
- mmsA : 24-26'
Claims (28)
- Fragment monocaténaire de l'ADN génomique de Streptococcus pneumoniae, caractérisé en ce qu'il comprend au moins une séquence nucléotidique présentant au moins 70% d'homologie avec au moins une séquence nucléotidique choisie parmi les séquences nucléotidiques SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4 et leurs séquences complémentaires respectives.
- Fragment selon la revendication 1, caractérisé en ce que la séquence nucléotidique dudit fragment présente au moins 85% d'homologie avec au moins une séquence nucléotidique choisie parmi les séquences nucléotidiques SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4 et leurs séquences complémentaires respectives.
- Sonde susceptible de s'hybrider spécifiquement avec l'ADN génomique de Streptococcus pneumoniae, caractérisée en ce qu'elle comprend une séquence nucléotidique qui, à la fois, présente au moins 70% d'homologie avec au moins une partie d'une séquence consensus de l'ADN génomique de Streptococcus pneumoniae choisie parmi les séquences nucléotidiques SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4 et leurs séquences complémentaires respectives, et s'hybride uniquement avec l'ADN génomique de Streptococcus pneumoniae.
- Sonde selon la revendication 3, caractérisée en ce que la séquence nucléotidique que ladite sonde comprend présente au moins 85% d'homologie avec au moins une partie de ladite séquence consensus.
- Sonde selon la revendication 3 ou 4, caractérisée en ce que la séquence nucléotidique que ladite sonde comprend, comporte au moins 12 nucléotides.
- Sonde selon l'une quelconque des revendications 3 à 5, caractérisée en ce qu'elle comprend la séquence nucléotidique, SEQ ID NO 3.
- Sonde selon la revendication 6, caractérisée en ce que ladite séquence nucléotidique SEQ ID NO 3 est flanquée à son extrémité 5' d'une séquence nucléotidique SEQ ID NO 2.
- Sonde selon la revendication 6, caractérisée en ce que ladite séquence nucléotidique SEQ ID NO 3 est flanquée à son extrémité 3' d'une séquence nucléotidique SEQ ID NO 4.
- Sonde selon la revendication 6, caractérisée en ce que la séquence nucléotidique SEQ ID NO 3 est répétée quatre fois de manière contiguë.
- Sonde selon la revendication 3, caractérisée en ce qu'elle est constituée par une séquence nucléotidique choisie parmi les séquences SEQ ID NO 5, SEQ ID NO 6 et SEQ ID NO 7.
- Sonde de capture d'ADN dénaturé caractérisée en ce qu'elle est choisie parmi les sondes selon l'une quelconque des revendications 3 à 10.
- Sonde selon l'une quelconque des revendications 1 à 10, caractérisée en ce qu'elle est marquée au moyen d'un marqueur choisi parmi des isotopes radioactifs, des enzymes choisis parmi la péroxydase et la phosphatase alcaline et ceux susceptibles d'hydrolyser un substrat chromogène, fluorigène ou luminescent, des composés chimiques chromophores, des composés chromogènes, fluorigènes ou luminescents, des analogues de bases nucléotidiques, et la biotine.
- Sonde de détection caractérisée en ce qu'elle est choisie parmi les sondes selon la revendication 12.
- Amorce spécifique pour l'amplification par la polymérisation de l'ADN génomique de Streptococcus pneumoniae, caractérisée en ce qu'elle comprend une séquence nucléotidique qui, à la fois, présente au moins 70% d'homologie avec au moins une partie d'une séquence consensus de l'ADN génomique de Streptococcus pneumoniae choisie parmi les séquences nucléotidiques SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4 et leurs séquences complémentaires respectives, et s'hybride uniquement avec l'ADN génomique de Streptococcus pneumoniae.
- Amorce selon la revendication 14, caractérisée en ce qu'elle est choisie parmi les amorces répondant aux séquences nucléotidiques respectivement SEQ ID NO 8 à SEQ ID NO 21.
- Couple d'amorces caractérisé en ce qu'il comprend au moins une amorce choisie parmi les amorces selon la revendication 15.
- Couple d'amorces selon la revendication 16, caractérisé en ce qu'il est choisi parmi les couples d'amorces constitués par une amorce répondant à la séquence nucléotidique SEQ ID NO 8 et une sonde répondant à l'une quelconque des séquences nucléotidiques SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 19 et SEQ ID NO 21 ou parmi les couples d'amorces constitués par une amorce répondant à la séquence nucléotidique SEQ ID NO 10 et une amorce répondant à l'une quelconque des séquences nucléotidiques SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 19 et SEQ ID NO 21.
- Réactif pour détecter sélectivement Streptococcus pneumoniae dans un échantillon biologique, caractérisé en ce qu'il comprend au moins une sonde selon l'une quelconque des revendications 3 à 10.
- Réactif selon la revendication 18, caractérisé en ce qu'il comprend une sonde selon la revendication 11.
- Réactif selon la revendication 19, caractérisé en ce qu'il comprend une sonde selon la revendication 12.
- Réactif selon la revendication 18, caractérisé en ce que la sonde est en milieu liquide ou fixée sur un support solide directement ou indirectement.
- Réactif selon la revendication 21, caractérisé en ce que ledit support est choisi parmi les polystyrènes, les copolymères styrène-butadiène, les copolymères styrène-butadiène en mélange avec des polystyrènes, des polypropylènes, des polycarbonates, des copolymères polystyrène-acrylonitrile, des copolymères styrène-méthylméthacrylate de méthyle, parmi les fibres synthétiques et naturelles, parmi les polysaccharides et les dérivés de la cellulose.
- Réactif selon la revendication 18, caractérisé en ce qu'il comprend en outre au moins une amorce selon la revendication 14 ou 15.
- Réactif selon la revendication 23, caractérisé en ce qu'il comprend un couple d'amorces selon la revendication 16 ou 17.
- Procédé de détection sélective de Streptococcus pneumoniae dans un échantillon biologique caractérisé en ce qu'on expose l'ADN génomique des bactéries contenues dans ledit échantillon, sous forme de fragments monocaténaires, à une sonde selon l'une quelconque des revendications 3 à 10 ou selon les revendications 12 et 13, et on détecte les zones d'hybridation avec ladite sonde.
- Procédé selon la revendication 25, caractérisé en ce qu'on expose l'ADN génomique des bactéries, à une sonde de détection selon la revendication 13.
- Procédé selon la revendication 26, caractérisé en ce qu'avant d'exposer l'ADN génomique des bactéries à ladite sonde de détection, on l'expose à une sonde de capture selon la revendication 11.
- Procédé selon lune quelconque des revendication 25 à 27, caractérisé en ce que avant d'exposer lesdits fragments à ladite sonde, on réalise une amplification de l'ADN génomique de Streptococcus pneumoniae, en présence d'un système enzymatique adapté et au moins une amorce selon la revendication 14 ou 15 ou un couple d'amorces selon la revendication 16 ou 17.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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FR9201655A FR2687168B1 (fr) | 1992-02-10 | 1992-02-10 | Fragment de l'adn genomique de streptococcus pneumoniae, sonde d'hybridation, amorce d'amplification, reactif et procede de detection de streptococcus pneumoniae. |
FR9201655 | 1992-02-10 |
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EP0577523A1 EP0577523A1 (fr) | 1994-01-05 |
EP0577523B1 true EP0577523B1 (fr) | 1998-04-22 |
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DE (1) | DE69318072T2 (fr) |
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US20020055101A1 (en) | 1995-09-11 | 2002-05-09 | Michel G. Bergeron | Specific and universal probes and amplification primers to rapidly detect and identify common bacterial pathogens and antibiotic resistance genes from clinical specimens for routine diagnosis in microbiology laboratories |
US6001564A (en) * | 1994-09-12 | 1999-12-14 | Infectio Diagnostic, Inc. | Species specific and universal DNA probes and amplification primers to rapidly detect and identify common bacterial pathogens and associated antibiotic resistance genes from clinical specimens for routine diagnosis in microbiology laboratories |
JP3571378B2 (ja) | 1994-09-30 | 2004-09-29 | 扶桑薬品工業株式会社 | 感染症診断用プローブ |
US5994066A (en) * | 1995-09-11 | 1999-11-30 | Infectio Diagnostic, Inc. | Species-specific and universal DNA probes and amplification primers to rapidly detect and identify common bacterial pathogens and associated antibiotic resistance genes from clinical specimens for routine diagnosis in microbiology laboratories |
US20100267012A1 (en) | 1997-11-04 | 2010-10-21 | Bergeron Michel G | Highly conserved genes and their use to generate probes and primers for detection of microorganisms |
US20030049636A1 (en) | 1999-05-03 | 2003-03-13 | Bergeron Michel G. | Species-specific, genus-specific and universal DNA probes and amplification primers to rapidly detect and identify common bacterial and fungal pathogens and associated antibiotic resistance genes from clinical specimens for diagnosis in microbiology laboratories |
US6270762B1 (en) | 1999-02-26 | 2001-08-07 | Smithkline Beecham Corporation | tdk |
BR0014370A (pt) | 1999-09-28 | 2002-11-05 | Infectio Diagnostic Inc | Genes altamente conservados e seu uso para gerar sondas de ácido nucléico e preparadores de amplificação especìficos em espécie, especìfico em gênero, especìficos em famìlia, especìficos em grupo e universais para rapidamente detectar e identificar microorganismos algais, amebianos, bacterianos, fúngicos e parasitas de espécimes clìnicos para diagnose |
EP2258842A1 (fr) * | 2001-04-16 | 2010-12-08 | Wyeth Holdings Corporation | Cadres de lecture ouverts de streptococcus pneumoniae codant pour des antigènes polypeptidiques, et leurs utilisations |
DE10136656A1 (de) * | 2001-07-27 | 2003-02-13 | Mwg Biotech Ag | Biochip und Verfahren für die Ermittlung von Sondensequenzen für einen Biochip |
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CA1256005A (fr) * | 1983-09-26 | 1989-06-20 | W. Peter Hansen | Methode d'hybridation sandwich pour la detection d'acides nucleiques |
US4683195A (en) * | 1986-01-30 | 1987-07-28 | Cetus Corporation | Process for amplifying, detecting, and/or-cloning nucleic acid sequences |
EP0305145A3 (fr) * | 1987-08-24 | 1990-05-02 | Ortho Diagnostic Systems Inc. | Méthodes et sondes pour détecter des acides nucléiques |
NL9002157A (nl) * | 1989-11-27 | 1991-06-17 | U Gene Research Bv | Dna fragmenten en daarop gebaseerde dna-probes en primers. |
FR2663040B1 (fr) * | 1990-06-11 | 1995-09-15 | Bio Merieux | Procede de detection d'une sequence nucleotidique selon la technique d'hybridation sandwich. |
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- 1992-02-10 FR FR9201655A patent/FR2687168B1/fr not_active Expired - Fee Related
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1993
- 1993-02-10 EP EP93420061A patent/EP0577523B1/fr not_active Expired - Lifetime
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Non-Patent Citations (1)
Title |
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J.Bacteriol.,vol.171,1989,pp.5332-5338,(Prudhome et al.). * |
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Publication number | Publication date |
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US5776691A (en) | 1998-07-07 |
EP0577523A1 (fr) | 1994-01-05 |
DE69318072T2 (de) | 1998-09-03 |
FR2687168B1 (fr) | 1994-03-25 |
FR2687168A1 (fr) | 1993-08-13 |
DE69318072D1 (de) | 1998-05-28 |
US5770362A (en) | 1998-06-23 |
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